Burn pit-related smoke causes developmental and behavioral toxicity in zebrafish: Influence of material type and emissions chemistry.

Combustion of mixed materials during open air burning of refuse or structural fires in the wildland urban interface produces emissions that worsen air quality, contaminate rivers and streams, and cause poor health outcomes including developmental effects. The zebrafish, a freshwater fish, is a useful model for quickly screening the toxicological and developmental effects of agents in such species and elicits biological responses that are often analogous and predictive of responses in mammals. The purpose of this study was to compare the developmental toxicity of smoke derived from the burning of 5 different burn pit-related material types (plywood, cardboard, plastic, a mixture of the three, and the mixture plus diesel fuel as an accelerant) in zebrafish larvae. Larvae were exposed to organic extracts of increasing concentrations of each smoke 6-to-8-hr post fertilization and assessed for morphological and behavioral toxicity at 5 days post fertilization. To examine chemical and biological determinants of toxicity, responses were related to emissions concentrations of polycyclic hydrocarbons (PAH). Emissions from plastic and the mixture containing plastic caused the most pronounced developmental effects, including mortality, impaired swim bladder inflation, pericardial edema, spinal curvature, tail kinks, and/or craniofacial deformities, although all extracts caused concentration-dependent effects. Plywood, by contrast, altered locomotor responsiveness to light changes to the greatest extent. Some morphological and behavioral responses correlated strongly with smoke extract levels of PAHs including 9-fluorenone. Overall, the findings suggest that material type and emissions chemistry impact the severity of zebrafish developmental toxicity responses to burn pit-related smoke.

Influence of Methylene Blue or Dimethyl Sulfoxide on Larval Zebrafish Development and Behavior.

The use of larval zebrafish developmental testing and assessment, specifically larval zebrafish locomotor activity, has been recognized as a higher throughput testing strategy to identify developmentally toxic and neurotoxic chemicals. There are, however, no standardized protocols for this type of assay, which could result in confounding variables being overlooked. Two chemicals commonly employed during early-life stage zebrafish assays, methylene blue (antifungal agent) and dimethyl sulfoxide (DMSO, a commonly used vehicle) have been reported to affect the morphology and behavior of freshwater fish. In this study, we conducted developmental toxicity (morphology) and neurotoxicity (behavior) assessments of commonly employed concentrations for both chemicals (0.6-10.0 µM methylene blue; 0.3%-1.0% v/v DMSO). A light-dark transition behavioral testing paradigm was applied to morphologically normal, 6 days postfertilization (dpf) zebrafish larvae kept at 26°C. Additionally, an acute DMSO challenge was administered based on early-life stage zebrafish assays typically used in this research area. Results from developmental toxicity screens were similar between both chemicals with no morphological abnormalities detected at any of the concentrations tested. However, neurodevelopmental results were mixed between the two chemicals of interest. Methylene blue resulted in no behavioral changes up to the highest concentration tested, 10.0 µM. By contrast, DMSO altered larval behavior following developmental exposure at concentrations as low as 0.5% (v/v) and exhibited differential concentration-response patterns in the light and dark photoperiods. These results indicate that developmental DMSO exposure can affect larval zebrafish locomotor activity at routinely used concentrations in developmental neurotoxicity assessments, whereas methylene blue does not appear to be developmentally or neurodevelopmentally toxic to larval zebrafish at routinely used concentrations. These results also highlight the importance of understanding the influence of experimental conditions on larval zebrafish locomotor activity that may ultimately confound the interpretation of results.

Inconsistencies in variable reporting and methods in larval zebrafish behavioral assays.

New approaches in developmental neurotoxicity (DNT) screening are needed due to the tens of thousands of chemicals requiring hazard assessments. Zebrafish (Danio rerio) are an alternative vertebrate model for DNT testing, but without a standardized protocol for larval behavioral assays, comparison of results among laboratories is challenging. To evaluate the congruence of protocols across laboratories, we conducted a literature review of DNT studies focusing on larval zebrafish behavior assays and cataloged experimental design consistencies. Our review focused on 51 unique method variables in publications where chemical exposure occurred in early development and subsequent larval locomotor evaluation focused on assays that included a light/dark photoperiod transition. We initially identified 94 publications, but only 31 exclusively met our inclusion criteria which focused on parameters that are important to an assay employed by our laboratory. No publication reported 100% of the targeted variables; only 51 to 86% of those variables were reported in the reviewed publications, with some aspects of the experimental design consistent among laboratories. However, no protocol was exactly the same for any two publications. Many of these variables had more than one parameter/design reported, highlighting the inconsistencies among methods. Overall, there is not only a strong need for the development of a standardized testing protocol for larval zebrafish locomotor assays, but there is also a need for a standardized protocol for reporting experimental variables in the literature. Here we include an extensive guideline checklist for conducting larval zebrafish developmental behavior assays.

Discovery of 1'-(1-phenylcyclopropane-carbonyl)-3H-spiro[isobenzofuran-1,3'-pyrrolidin]-3-one as a novel steroid mimetic scaffold for the potent and tissue-specific inhibition of 11ß-HSD1 using a scaffold-hopping approach.

11ß-hydroxysteroid dehydrogenase 1 (11ß-HSD1) has been identified as the primary enzyme responsible for the activation of hepatic cortisone to cortisol in specific peripheral tissues resulting in the concomitant antagonism of insulin action within these tissues. Dysregulation of 11ß-HSD1, particularly in adipose tissues, has been associated with metabolic syndrome and type 2 diabetes mellitus. Therefore, inhibition of 11ß-HSD1 with a small nonsteroidal molecule is therapeutically desirable. Implementation of a scaffold-hopping approach revealed a three-point pharmacophore for 11ß-HSD1 that was utilized to design a steroid mimetic scaffold. Reiterative optimization provided valuable insight into the bioactive conformation of our novel scaffold and led to the discovery of INCB13739. Clinical evaluation of INCB13739 confirmed for the first time that tissue-specific inhibition of 11ß-HSD1 in patients with type 2 diabetes mellitus was efficacious in controlling glucose levels and reducing cardiovascular risk factors.

Developmental Neurotoxicity and Behavioral Screening in Larval Zebrafish with a Comparison to Other Published Results.

With the abundance of chemicals in the environment that could potentially cause neurodevelopmental deficits, there is a need for rapid testing and chemical screening assays. This study evaluated the developmental toxicity and behavioral effects of 61 chemicals in zebrafish (Danio rerio) larvae using a behavioral Light/Dark assay. Larvae (n = 16-24 per concentration) were exposed to each chemical (0.0001-120 µM) during development and locomotor activity was assessed. Approximately half of the chemicals (n = 30) did not show any gross developmental toxicity (i.e., mortality, dysmorphology or non-hatching) at the highest concentration tested. Twelve of the 31 chemicals that did elicit developmental toxicity were toxic at the highest concentration only, and thirteen chemicals were developmentally toxic at concentrations of 10 µM or lower. Eleven chemicals caused behavioral effects; four chemicals (6-aminonicotinamide, cyclophosphamide, paraquat, phenobarbital) altered behavior in the absence of developmental toxicity. In addition to screening a library of chemicals for developmental neurotoxicity, we also compared our findings with previously published results for those chemicals. Our comparison revealed a general lack of standardized reporting of experimental details, and it also helped identify some chemicals that appear to be consistent positives and negatives across multiple laboratories.

Zebrafish Locomotor Responses Reveal Irritant Effects of Fine Particulate Matter Extracts and a Role for TRPA1.

Exposure to fine particulate matter (PM) air pollution causes adverse cardiopulmonary outcomes. Yet, the limited capacity to readily identify contributing PM sources and associated PM constituents in any given ambient air shed impedes risk assessment efforts. The health effects of PM have been attributed in part to its capacity to elicit irritant responses. A variety of chemicals trigger irritant behavior responses in zebrafish that can be easily measured. The purposes of this study were to examine the utility of zebrafish locomotor responses in the toxicity assessment of fine PM and its chemical fractions and uncover mechanisms of action. Locomotor responses were recorded in 6-day-old zebrafish exposed for 60 min in the dark at 26 °C to the extractable organic matter of a compressor-generated diesel exhaust PM (C-DEP) and 4 of its fractions (F1-F4) containing varying chemical classes of increasing polarity. The role of the transient receptor potential (TRP) cation channel TRPA1, a chemical sensor in mammals and zebrafish, in locomotor responses to C-DEP, was also examined. Acrolein, an environmental irritant and known activator of TRPA1, and all extracts induced concentration-dependent locomotor responses whose potencies ranked as follows: polar F3 > weakly polar F2 > C-DEP > highly polar F4 > nonpolar F1, indicating that polar and weakly polar fractions that included nitro- and oxy-polyaromatic hydrocarbons (PAHs), drove C-DEP responses. Irritant potencies in fish positively correlated with mutagenic potencies of the same extracts in strains of Salmonella sensitive to nitro- and oxy-PAHs, further implicating these chemical classes in the zebrafish responses to C-DEP. Pharmacologic inhibition of TRPA1 blocked locomotor responses to acrolein and the extracts. Taken together, these data indicate that the zebrafish locomotor assay may help expedite toxicity screening of fine PM sources, identify causal chemical classes, and uncover plausible biological mechanisms.

The efficacy and safety of continued hydroxycarbamide therapy versus switching to ruxolitinib in patients with polycythaemia vera: a randomized, double-blind, double-dummy, symptom study (RELIEF).

The randomized, double-blind, double-dummy, phase 3b RELIEF trial evaluated polycythaemia vera (PV)-related symptoms in patients who were well controlled with a stable dose of hydroxycarbamide (also termed hydroxyurea) but reported PV-related symptoms. Patients were randomized 1:1 to ruxolitinib 10 mg BID (n = 54) or hydroxycarbamide (prerandomization dose/schedule; n = 56); crossover to ruxolitinib was permitted after Week 16. The primary endpoint, ≥50% improvement from baseline in myeloproliferative neoplasm -symptom assessment form total symptom score cytokine symptom cluster (TSS-C; sum of tiredness, itching, muscle aches, night sweats, and sweats while awake) at Week 16, was achieved by 43·4% vs. 29·6% of ruxolitinib- and hydroxycarbamide-treated patients, respectively (odds ratio, 1·82; 95% confidence interval, 0·82-4·04; P = 0·139). The primary endpoint was achieved by 34% of a subgroup who maintained their hydroxycarbamide dose from baseline to Weeks 13-16. In a post hoc analysis, the primary endpoint was achieved by more patients with stable screening-to-baseline TSS-C scores (ratio ≤ 2) receiving ruxolitinib than hydroxycarbamide (47·4% vs. 25·0%; P = 0·0346). Ruxolitinib treatment after unblinding was associated with continued symptom score improvements. Adverse events were primarily grades 1/2 with no unexpected safety signals. Ruxolitinib was associated with a nonsignificant trend towards improved PV-related symptoms versus hydroxycarbamide, although an unexpectedly large proportion of patients who maintained their hydroxycarbamide dose reported symptom improvement.

Primary analysis of a phase II open-label trial of INCB039110, a selective JAK1 inhibitor, in patients with myelofibrosis.

Combined Janus kinase 1 (JAK1) and JAK2 inhibition therapy effectively reduces splenomegaly and symptom burden related to myelofibrosis but is associated with dose-dependent anemia and thrombocytopenia. In this open-label phase II study, we evaluated the efficacy and safety of three dose levels of INCB039110, a potent and selective oral JAK1 inhibitor, in patients with intermediate- or high-risk myelofibrosis and a platelet count ≥50×109/L. Of 10, 45, and 32 patients enrolled in the 100 mg twice-daily, 200 mg twice-daily, and 600 mg once-daily cohorts, respectively, 50.0%, 64.4%, and 68.8% completed week 24. A ≥50% reduction in total symptom score was achieved by 35.7% and 28.6% of patients in the 200 mg twice-daily cohort and 32.3% and 35.5% in the 600 mg once-daily cohort at week 12 (primary end point) and 24, respectively. By contrast, two patients (20%) in the 100 mg twice-daily cohort had ≥50% total symptom score reduction at weeks 12 and 24. For the 200 mg twice-daily and 600 mg once-daily cohorts, the median spleen volume reductions at week 12 were 14.2% and 17.4%, respectively. Furthermore, 21/39 (53.8%) patients who required red blood cell transfusions during the 12 weeks preceding treatment initiation achieved a ≥50% reduction in the number of red blood cell units transfused during study weeks 1-24. Only one patient discontinued for grade 3 thrombocytopenia. Non-hematologic adverse events were largely grade 1 or 2; the most common was fatigue. Treatment with INCB039110 resulted in clinically meaningful symptom relief, modest spleen volume reduction, and limited myelosuppression.

Acute and developmental behavioral effects of flame retardants and related chemicals in zebrafish.

As polybrominated diphenyl ethers are phased out, numerous compounds are emerging as potential replacement flame retardants for use in consumer and electronic products. Little is known, however, about the neurobehavioral toxicity of these replacements. This study evaluated the neurobehavioral effects of acute or developmental exposure to t-butylphenyl diphenyl phosphate (BPDP), 2-ethylhexyl diphenyl phosphate (EHDP), isodecyl diphenyl phosphate (IDDP), isopropylated phenyl phosphate (IPP), tricresyl phosphate (TMPP; also abbreviated TCP), triphenyl phosphate (TPHP; also abbreviated TPP), tetrabromobisphenol A (TBBPA), tris (2-chloroethyl) phosphate (TCEP), tris (1,3-dichloroisopropyl) phosphate (TDCIPP; also abbreviated TDCPP), tri-o-cresyl phosphate (TOCP), and 2,2-,4,4'-tetrabromodiphenyl ether (BDE-47) in zebrafish (Danio rerio) larvae. Larvae (n≈24 per dose per compound) were exposed to test compounds (0.4-120 µM) at sub-teratogenic concentrations either developmentally or acutely, and locomotor activity was assessed at 6 days post fertilization. When given developmentally, all chemicals except BPDP, IDDP and TBBPA produced behavioral effects. When given acutely, all chemicals produced behavioral effects, with TPHP, TBBPA, EHDP, IPP, and BPDP eliciting the most effects at the most concentrations. The results indicate that these replacement flame retardants may have developmental or pharmacological effects on the vertebrate nervous system.

Randomized, Double-Blind, Phase II Study of Ruxolitinib or Placebo in Combination With Capecitabine in Patients With Metastatic Pancreatic Cancer for Whom Therapy With Gemcitabine Has Failed.

PURPOSE: Patients with advanced pancreatic adenocarcinoma have a poor prognosis and limited second-line treatment options. Evidence suggests a role for the Janus kinase (JAK)/signal transducer and activator of transcription pathway in the pathogenesis and clinical course of pancreatic cancer. PATIENTS AND METHODS: In this double-blind, phase II study, patients with metastatic pancreatic cancer who had experienced treatment failure with gemcitabine were randomly assigned 1:1 to the JAK1/JAK2 inhibitor ruxolitinib (15 mg twice daily) plus capecitabine (1,000 mg/m(2) twice daily) or placebo plus capecitabine. The primary end point was overall survival (OS); secondary end points included progression-free survival, clinical benefit response, objective response rate, and safety. Prespecified subgroup analyses evaluated treatment heterogeneity and efficacy in patients with evidence of inflammation. RESULTS: In the intent-to-treat population (ruxolitinib, n = 64; placebo, n = 63), the hazard ratio was 0.79 (95% CI, 0.53 to 1.18; P = .25) for OS and was 0.75 (95% CI, 0.52 to 1.10; P = .14) for progression-free survival. In a prespecified subgroup analysis of patients with inflammation, defined by serum C-reactive protein levels greater than the study population median (ie, 13 mg/L), OS was significantly greater with ruxolitinib than with placebo (hazard ratio, 0.47; 95% CI, 0.26 to 0.85; P = .011). Prolonged survival in this subgroup was supported by post hoc analyses of OS that categorized patients by the modified Glasgow Prognostic Score, a systemic inflammation-based prognostic system. Grade 3 or greater adverse events were observed with similar frequency in the ruxolitinib (74.6%) and placebo (81.7%) groups. Grade 3 or greater anemia was more frequent with ruxolitinib (15.3%; placebo, 1.7%). CONCLUSION: Ruxolitinib plus capecitabine was generally well tolerated and may improve survival in patients with metastatic pancreatic cancer and evidence of systemic inflammation.

Developmental exposure to organophosphate flame retardants elicits overt toxicity and alters behavior in early life stage zebrafish (Danio rerio).

Organophosphate flame retardants (OPFRs) are common replacements for the phased-out polybrominated diphenyl ethers (PBDEs) and have been detected at high concentrations in environmental samples. OPFRs are structurally similar to organophosphate pesticides and may adversely affect the developing nervous system. This study evaluated the overt toxicity, uptake, and neurobehavioral effects of tris (1,3-dichloro-2-propyl) phosphate (TDCPP), tris (2-chloroethyl) phosphate (TCEP), tris (1-chloro-2-propyl) phosphate (TCPP), and tris (2,3-dibromopropyl) phosphate (TDBPP) in early life stage zebrafish. Chlorpyrifos was used as a positive control. For overt toxicity and neurobehavioral assessments, zebrafish were exposed from 0 to 5 days postfertilization (dpf). Hatching, death, or malformations were evaluated daily. Teratogenic effects were scored by visual examination on 6 dpf. To evaluate uptake and metabolism, zebrafish were exposed to 1 µM of each organophosphate (OP) flame retardant and collected on 1 and 5 dpf to monitor accumulation. Larval swimming activity was measured in 6 dpf larvae to evaluate neurobehavioral effects of exposures below the acute toxicity threshold. TDBPP elicited the greatest toxicity at >1 µM. TDCPP and chlorpyrifos were overtly toxic at concentrations ≥10 µM, TCEP, and TCPP were not overtly toxic at the doses tested. Tissue concentrations increased with increasing hydrophobicity of the parent chemical after 24 h exposures. TDCPP and TDBPP and their respective metabolites were detected in embryos on 5 dpf. For all chemicals tested, developmental exposures that were not overtly toxic significantly altered larval swimming activity. These data indicate that OPFRs adversely affect development of early life stage zebrafish.

Comparison of placebo and best available therapy for the treatment of myelofibrosis in the phase 3 COMFORT studies.

Prior to Janus kinase inhibitors, available therapies for myelofibrosis were generally supportive and did not improve survival. This analysis compares efficacy outcomes of patients with myelofibrosis in the control arms (placebo [n=154] and best available therapy [n=73]) from the two phase 3 COntrolled MyeloFibrosis study with ORal JAK inhibitor Treatment (COMFORT) studies. Spleen volume was assessed by magnetic resonance imaging/computed tomography at baseline and every 12 weeks through week 72; spleen length was assessed by palpation at each study visit. Health-related quality of life and symptoms were assessed using the European Organisation for Research and Treatment of Cancer Quality of Life Questionnaire-Core 30 Items at baseline and in weeks 4, 8, 12, 16 and 24 in COMFORT-I and in weeks 8, 16, 24 and 48 in COMFORT-II. The demographic and baseline characteristics were similar between the control arms of the two studies. One patient who received placebo and no patients who received best available therapy had a ≥35% reduction in spleen volume from baseline at week 24. At 24 weeks, neither placebo nor best available therapy had produced clinically meaningful changes in global quality of life or symptom scales. Non-hematologic adverse events were mostly grade 1/2; the most frequently reported adverse events in each group were abdominal pain, fatigue, peripheral edema and diarrhea. These data suggest that non-Janus kinase inhibitor therapies provide little improvement in splenomegaly, symptoms or quality of life as compared with placebo. Both COMFORT-I (NCT00952289) and COMFORT-II (NCT00934544) studies have been appropriately registered with clinicaltrials.gov.

Three-year efficacy, safety, and survival findings from COMFORT-II, a phase 3 study comparing ruxolitinib with best available therapy for myelofibrosis.

Ruxolitinib is a potent Janus kinase (JAK)1/JAK2 inhibitor that has demonstrated rapid reductions in splenomegaly and marked improvement in disease-related symptoms and quality of life in patients with myelofibrosis (MF). The present analysis reports the 3-year follow-up (median, 151 weeks) of the efficacy and safety of Controlled Myelofibrosis Study With Oral Janus-associated Kinase (JAK) Inhibitor Treatment-II (the COMFORT-II Trial), comparing ruxolitinib with the best available therapy (BAT) in 219 patients with intermediate-2 and high-risk MF. In the ruxolitinib arm, with continued therapy, spleen volume reductions of ≥35% by magnetic resonance imaging (equivalent to approximately 50% reduction by palpation) were sustained for at least 144 weeks, with the probability of 50% (95% confidence interval [CI], 36-63) among patients achieving such degree of response. At the time of this analysis, 45% of the patients randomized to ruxolitinib remained on treatment. Ruxolitinib continues to be well tolerated. Anemia and thrombocytopenia were the main toxicities, but they were generally manageable, improved over time, and rarely led to treatment discontinuation (1% and 3.6% of patients, respectively). No single nonhematologic adverse event led to definitive ruxolitinib discontinuation in more than 1 patient. Additionally, patients randomized to ruxolitinib showed longer overall survival than those randomized to BAT (hazard ratio, 0.48; 95% CI, 0.28-0.85; log-rank test, P = .009).

Health-related quality of life and symptoms in patients with myelofibrosis treated with ruxolitinib versus best available therapy.

Patients with myelofibrosis (MF) have significant debilitating symptoms, physical disabilities, and poor health-related quality of life (HRQoL). Here, we report post-hoc analyses of the impact of ruxolitinib, a potent and selective JAK1 and JAK2 inhibitor, on disease-related symptoms and HRQoL in MF patients from the large phase 3 COMFORT-II study (N = 219). During the follow-up period of 48 weeks, HRQoL and MF-associated symptoms improved from baseline for ruxolitinib-treated patients but remained the same or worsened for best available therapy (BAT)-treated patients. Based on the European Organization for Research and Treatment of Cancer QoL Questionnaire core 30 items (EORTC QLQ-C30), treatment-induced differences in physical and role functioning, fatigue, and appetite loss significantly favoured ruxolitinib versus BAT from week 8 (P < 0·05) up to week 48 (P < 0·05). Ruxolitinib resulted in significantly higher response rates in global health status/QoL and Functional Assessment of Cancer Therapy-Lymphoma (FACT-Lym) summary scores versus BAT at most time points (P < 0·05). Significant improvements in the Lymphoma subscale (including symptoms of pain, fever, itching, fatigue, weight loss, loss of appetite, and other patient concerns), FACT-General, FACT-Lym trial outcome index, and FACT-Lym total were also observed with ruxolitinib versus BAT starting at week 8 and continuing thereafter. Overall, these data demonstrated that ruxolitinib improved HRQoL in MF patients and further support the use of ruxolitinib for the treatment of symptomatic MF.

JAK inhibition with ruxolitinib versus best available therapy for myelofibrosis.

BACKGROUND: Treatment options for myelofibrosis are limited. We evaluated the efficacy and safety of ruxolitinib, a potent and selective Janus kinase (JAK) 1 and 2 inhibitor, as compared with the best available therapy, in patients with myelofibrosis. METHODS: We assigned 219 patients with intermediate-2 or high-risk primary myelofibrosis, post-polycythemia vera myelofibrosis, or post-essential thrombocythemia myelofibrosis to receive oral ruxolitinib or the best available therapy. The primary end point and key secondary end point of the study were the percentage of patients with at least a 35% reduction in spleen volume at week 48 and at week 24, respectively, as assessed with the use of magnetic resonance imaging or computed tomography. RESULTS: A total of 28% of the patients in the ruxolitinib group had at least a 35% reduction in spleen volume at week 48, as compared with 0% in the group receiving the best available therapy (P<0.001); the corresponding percentages at week 24 were 32% and 0% (P<0.001). At 48 weeks, the mean palpable spleen length had decreased by 56% with ruxolitinib but had increased by 4% with the best available therapy. The median duration of response with ruxolitinib was not reached, with 80% of patients still having a response at a median follow-up of 12 months. Patients in the ruxolitinib group had an improvement in overall quality-of-life measures and a reduction in symptoms associated with myelofibrosis. The most common hematologic abnormalities of grade 3 or higher in either group were thrombocytopenia and anemia, which were managed with a dose reduction, interruption of treatment, or transfusion. One patient in each group discontinued treatment owing to thrombocytopenia, and none discontinued owing to anemia. Nonhematologic adverse events were rare and mostly grade 1 or 2. Two cases of acute myeloid leukemia were reported with the best available therapy. CONCLUSIONS: Continuous ruxolitinib therapy, as compared with the best available therapy, was associated with marked and durable reductions in splenomegaly and disease-related symptoms, improvements in role functioning and quality of life, and modest toxic effects. An influence on overall survival has not yet been shown. (Funded by Novartis Pharmaceuticals; ClinicalTrials.gov number, NCT00934544.).

Developmental exposure to valproate and ethanol alters locomotor activity and retino-tectal projection area in zebrafish embryos.

Given the minimal developmental neurotoxicity data available for the large number of new and existing chemicals, there is a critical need for alternative methods to identify and prioritize chemicals for further testing. We outline a developmental neurotoxicity screening approach using zebrafish embryos. Embryos were exposed to nominal concentrations of either valproate or ethanol then examined for lethality, malformation, nervous system structure and locomotor activity. Developmental valproate exposure caused locomotor activity changes at concentrations that did not result in malformations and showed a concentration-dependent decrease in retino-tectal projection area in the optic tectum. Developmental ethanol exposure also affected retino-tectal projection area at concentrations below those concentrations causing malformations. As both valproate and ethanol are known human developmental neurotoxicants, these results add to the growing body of evidence showing the potential utility of zebrafish in screening compounds for mammalian developmental neurotoxicity.

The metamorphosis of supernova SN 2008D/XRF 080109: a link between supernovae and GRBs/hypernovae.

The only supernovae (SNe) to show gamma-ray bursts (GRBs) or early x-ray emission thus far are overenergetic, broad-lined type Ic SNe (hypernovae, HNe). Recently, SN 2008D has shown several unusual features: (i) weak x-ray flash (XRF), (ii) an early, narrow optical peak, (iii) disappearance of the broad lines typical of SN Ic HNe, and (iv) development of helium lines as in SNe Ib. Detailed analysis shows that SN 2008D was not a normal supernova: Its explosion energy (E approximately 6x10(51) erg) and ejected mass [ approximately 7 times the mass of the Sun (M(middle dot in circle))] are intermediate between normal SNe Ibc and HNe. We conclude that SN 2008D was originally a approximately 30 M(middle dot in circle) star. When it collapsed, a black hole formed and a weak, mildly relativistic jet was produced, which caused the XRF. SN 2008D is probably among the weakest explosions that produce relativistic jets. Inner engine activity appears to be present whenever massive stars collapse to black holes.

Temporal organization of phytoplankton communities linked to physical forcing.

The performance of individual phytoplankton species is strongly governed by the thermal stratification's impact on vertical mixing within the water column, which alters the position of phytoplankton relative to nutrients and light. The present study documents shifts in phytoplankton structure and vertical positioning that have accompanied intensified long-term stratification in a natural ecosystem. Ordination analysis is used to extract gradients in phytoplankton composition in Lake Tahoe, an extremely nutrient-poor lake, over a 23-year period of records. Community structure in the 1980s was associated most strongly with resource availability (low nitrogen to phosphorus ratios, deeper euphotic zone depth), while intensified stratification dominated the phytoplankton structure since the late 1990s. Within diatoms, small-sized cells increased with reduced mixing, suggesting that suppressed turbulence provides them with a competitive advantage over large-sized cells. Among the morphologically diverse chlorophytes, filamentous and coenobial forms were favored under intensified stratification. The selection for small-sized diatoms is accompanied by a shoaling trend in their vertical position in the water column. In contrast, the motile flagellates displayed a deeper vertical positioning in recent years, indicating that optimal growth conditions shifted likely due to reduced upwelling of nutrients. As the thermal stratification of lakes and oceans is strongly linked to climate variables, the present study confirms that climate warming will alter phytoplankton structure and dynamics largely through effects on nutrient availability and sinking velocities. Intensified stratification should favor the expansion of small-sized species and species with the capability of buoyancy regulation, which may alter primary productivity, nutrient recycling, and higher trophic productivity.

CCR2 modulates inflammatory and metabolic effects of high-fat feeding.

The C-C motif chemokine receptor-2 (CCR2) regulates monocyte and macrophage recruitment and is necessary for macrophage-dependent inflammatory responses and the development of atherosclerosis. Although adipose tissue expression and circulating concentrations of CCL2 (also known as MCP1), a high-affinity ligand for CCR2, are elevated in obesity, the role of CCR2 in metabolic disorders, including insulin resistance, hepatic steatosis, and inflammation associated with obesity, has not been studied. To determine what role CCR2 plays in the development of metabolic phenotypes, we studied the effects of Ccr2 genotype on the development of obesity and its associated phenotypes. Genetic deficiency in Ccr2 reduced food intake and attenuated the development of obesity in mice fed a high-fat diet. In obese mice matched for adiposity, Ccr2 deficiency reduced macrophage content and the inflammatory profile of adipose tissue, increased adiponectin expression, ameliorated hepatic steatosis, and improved systemic glucose homeostasis and insulin sensitivity. In mice with established obesity, short-term treatment with a pharmacological antagonist of CCR2 lowered macrophage content of adipose tissue and improved insulin sensitivity without significantly altering body mass or improving hepatic steatosis. These data suggest that CCR2 influences the development of obesity and associated adipose tissue inflammation and systemic insulin resistance and plays a role in the maintenance of adipose tissue macrophages and insulin resistance once obesity and its metabolic consequences are established.